Enteroendocrine peptides regulate feeding behavior via controlling intestinal contraction of the silkworm <i>Bombyx mori</i>

Our previous study demonstrated that predominant feeding inhibitory effects were found in the crude extracts of foregut and midgut of the silkworm Bombyx mori larvae. To address the entero-intestinal control crucial for the regulation of insect feeding behavior, the present study identified and functionally characterized feeding inhibitory peptides from the midgut of B. mori larvae. Purification and structural analyses revealed that the predominant inhibitory factors in the crude extracts were allatotropin (AT) and GSRYamide after its C-terminal sequence. In situ hybridization revealed that AT and GSRYamide were expressed in enteroendocrine cells in the posterior and anterior midgut, respectively. Receptor screening using Ca2+-imaging technique showed that the B. mori neuropeptide G protein-coupled receptor (BNGR)-A19 and -A22 acted as GSRYamide receptors and BNGR-A5 acted as an additional AT receptor. Expression analyses of these receptors and the results of the peristaltic motion assay indicated that these peptides participated in the regulation of intestinal contraction. Exposure of pharynx and ileum to AT and GSRYamide inhibited spontaneous contraction in ad libitum-fed larvae, while exposure of pharynx to GSRYamide did not inhibit contraction in non-fed larvae, indicating that the feeding state changed their sensitivity to inhibitory peptides. These different responses corresponded to different expression levels of their receptors in the pharynx. In addition, injection of AT and GSRYamide decreased esophageal contraction frequencies in the melamine-treated transparent larvae. These findings strongly suggest that these peptides exert feeding inhibitory effects by modulating intestinal contraction in response to their feeding state transition, eventually causing feeding termination.我们前期研究发现，家蚕（Bombyx mori）幼虫前肠与中肠的粗提物具有显著的取食抑制活性。为阐明对昆虫取食行为调控至关重要的肠内调控机制，本研究从家蚕幼虫中肠中分离鉴定了取食抑制肽并对其功能进行了表征。纯化与结构分析结果显示，粗提物中的主要抑制因子为咽侧体刺激肽（allatotropin, AT）与C端序列为GSRYamide的肽（GSRYamide）。原位杂交（in situ hybridization）实验表明，AT与GSRYamide分别在中肠后部与前部的肠内分泌细胞中表达。采用钙离子成像（Ca2+-imaging）技术进行受体筛选发现，家蚕神经肽G蛋白偶联受体（BNGR）-A19与-A22为GSRYamide的受体，而BNGR-A5为AT的另一受体。对这些受体的表达分析以及蠕动运动实验结果表明，上述肽类参与了肠道收缩的调控。将咽与回肠暴露于AT与GSRYamide后，可抑制自由取食幼虫的自发性收缩；而将咽暴露于GSRYamide时，并未抑制未取食幼虫的收缩，这表明取食状态会改变组织对抑制肽的敏感性。这种差异响应与受体在咽部的不同表达水平相关。此外，向三聚氰胺处理后的透明幼虫体内注射AT与GSRYamide，可降低其食管收缩频率。这些研究结果强烈表明，上述肽类可通过响应取食状态转变调控肠道收缩，从而发挥取食抑制作用，最终导致取食终止。